Cloning, Prokaryotic Expression And Immune Function Analysis Of TAB1 Gene

The mitogen-activated protein kinases (MAPKs) are a family of serine/threonine kinases that regulate diverse biological processes. Transforming growth factor-β activated kinase-1 (TAK1) is also known as mitogen-activated protein kinase kinase kinase 7(MAP3K7), which was originally discovered as a serine/threonine kinase in the mitogen-activated protein kinase (MAPK) family. TAK1 mediates the activation of the nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), and p38 pathways in response to interleukin-1, tumor necrosis factor-a, and toll-like receptor agonists. The TAB1 (TAK1-binding protein) is a regulatory subunit of the protein kinase TAK1 and specifically activates the TAK1 kinase activity. Amphioxus, as a transitional species from invertebrates to vertebrates in evolutionary history, occupies the basal position of the chordate phylum and is an important reference to the evolution of vertebrate immunity. In order to further understand the function and evolution of TAB1, in this study, we cloned a TAB1 gene from Chinese amphioxus(Branchiostoma belcheri) (named as AmphiTAB1), and made a bioinformatics analysis on the sequence, as well as performed prokaryotic expression of the AmphiTAB1 protein. Quantitative RT-PCR analysis was used to detect the spatial and temporal expression of AmphiTAB1. The results are as follows:(1) A full-length amphioxus cDNA of 2281 bp was cloned by RT-PCR and RACE-PCR.The complete AmphiTAB1 cDNA contains an ORF of 1,659 bp nucleotides that encodes a 553 amino acid polypeptide, a 5’-UTR of 89 nucleotides and a 3’-UTR of 533 nucleotides with a consensus polyadenylation signal (AATAAA). An expression vector, pET-32a(+)-AmphiTAB1, was constructed by recombinant DNA technology. The expression of AmphiTAB1 protein was induced by addition of IPTG in E. coli for 6h at 26℃. The prokaryotic expression expressed a protein with a molecular mass of 62 kDa successfully. Western blot also showed the corresponding band in the lane of recombinant strains.(2) Bioinformatics analysis showed that AmphiTABl shared high sequence similarity with other identified and predicted TAB1 proteins. AmphiTAB1 gene has eight exons separated by seven introns. AmphiTAB 1 also contains a characteristic PP2Cc domain, which is highly conservative in TAB1 family members. Phylogenetic analysis indicated that the AmphiTAB1 gene was located between invertebrates and vertebrates, suggesting that the AmphiTABl gene is a member of the TAB1 gene family.(3) Quantitative RT-PCR was employed to quantify the expression pattern of AmphiTABl gene in different tissues including gills, hepatic cecum, intestine, muscles, notochord and gonad. The result showed that the AmphiTABl gene was constitutively expressed in all detected tissues. It is worth to note that the expression level of AmphiTABl gene was abundant in gonad, moderate in hepatic cecum, muscle and notochord, and low in gill and intestine. The AmphiTAB1 gene expression at different time points (2,4,6,8,10,12,24 and 48 h) after LPS stimulation was also measured using quantitative RT-PCR method. The peak of AmphiTABl mRNA transcripts was observed at 6 h post-injection, which was 2-fold to that in the control group (P<0.05).In conclusion, this study not only identified and characterized AmphiTAB1 for the first time in amphioxus, but also provided an insight into the evolution and innate immunity of TAB1 gene.